Activin and follistatin (FS) are novel peptides that coordinate growth and differentiation of basic tissues in vertebrates. Published results are compatible with a model in which activin functions as a ligand to induce cytodifferentiation, while FS, the activin-binding protein, prevents its function. The presence of activin and FS in granulosa cells (GC), implies a role in regulating normal physiological processes during follicle developmental; however, the physiological roles of FS and activin in follicle development are not known. In the present grant, we plan to use in vivo and in vitro systems to study the proposition that follicle selection requires coordinate expression of activin and FS to ensure proper timing of granulosa growth and differentiation, and that independent control (either over or under expression) of these genes leads to programmed cell death and atresia. Four specific aims have been formulated to identify how these genes are affected by normal selection and atresia, to discover at which level of their expression they are regulated by hormones and growth factors, ie. transcription and/or translation, and to determine their roles in the follicle regulation system. Aim #1:Determine whether there is coordinate or independent expression of FS and activin genes in GC of the same follicle during normal selection and atresia. In situ hybridization and immunocytochemistry will be used to assay for FS (288 and 315), and the activin/inhibin subunites (beta A, beta B and alpha) in GC of individual follicles throughout the estrous cycle. Aim #2: Determine the positive and negative regulatory molecules that control FS and activin expression in vitro. Rat GC cultured in serum free medium will be challenged with selectogenic (FSH, LH< PRL, E2), atretogenic (DHT, PGF 2 alpha GnRH, angiotensin II) and growth factors that amplify (IGF-I, TGFbeta) and attenuate (TGFalpha, BFGF, TNFalpha, IL-1) GC differentiation. Time course, dose response, reversibility and combination experiments are planned. Northern blot and Western ligand and immunoblotting will be used to assay FS and activin/inhibin subunits ( betaA, betaB and alpha). These studies will also focus on the effects of these molecules on cell growth and specific markers of differentiation, E2, P4 and 20 alpha DHP. Aim #3: Determine whether FS, activin and inhibin directly regulate their own expression in GC. These studies will proceed coordinately with Aim #2 and follow a similar experimental design. Aim #4: Examine the functional significance of FS and activin in folliculogenesis in vivo. Selection and atresia will be induced experimentally in vivo and the temporal pattern of FS and activin expression monitored by in situ hybridization and immunocytochemistry. To address the functions, we will inject recombinant activin and FS to determine their roles in inducing and reversing the atretic process in vivo. We anticipate the results of these experiments will generate new knowledge and understanding of the roles that FS and activin may play in normal follicle selection and atresia. Since these genes are expressed in human ovaries, our results could have clinical implications.